Pharmaceutical preparation for controlling pathogenic intestinal bacteria

ABSTRACT

A pharmaceutical preparation for controlling pathogenic microorganisms causing diarrhoea and other gastrointestinal troubles in man and in animals contains Streptococcus lactis strain LIa in at least one pharmaceutically acceptable carrier medium in which the microorganism retains its viability. The use of the preparation for controlling pathogenic microorganisms causing diarrhoea and other gastrointestinal infections in man and in animals is also described.

The present invention relates to a pharmaceutical preparation forcontrolling pathogenic intestinal bacteria in man and in animals, with aview to preventing and/or treating diarrhoea and other gastrointestinaltroubles.

BACKGROUND OF THE INVENTION

Diarrhoea and other gastrointestinal troubles caused by pathogenicmicroorganisms constitute a widespread health problem, which affects inparticular tourists travelling abroad, people having received cancertherapy involving antibiotics or radiation, and people working in daynurseries. Such pathogenic intestinal bacteria causing diarrhoea includeSalmonella, Shigella, Yersinia, E. coli, Pseudomonas, Clostridiumdificile and sordelli, Stafylococcus aureus and Campylobactus. Thesepathogenic microorganisms adhere to the mucous membranes of theintestine and upset the intestinal function, thereby causing diarrhoea.

It is well-known that microorganisms affect each other positively ornegatively by promoting or restraining each other's growth. Thisinterference phenomenon has above all been studied in the flora of theskin, the pharynx and the intestine of human beings. Although theunderlying causes are not fully known, it has been found that the normalbacterial flora is of considerable importance in the defence againstpathogenic bacteria. The bacteria of the normal flora inhibit invadingbacteria in many different ways, e.g. by producing antibiotic-likesubstances called bacteriocines. Unlike antibiotics, the bacteriocinesmostly have a very selective effect on a specific group of bacteriawithout influencing the remaining bacterial flora. Treating infectedpatients with such harmless bacteria of specific effect has been notonly discussed, but also tried to some extent. Naturally, the bacteriasupplied must be able to settle in the intestinal area where they are toproduce their effect. This is one of the ideas behind giving soured milkto patients whose intestinal flora is disturbed, and who suffer fromsevere diarrhoea after treatment with antibiotics. However, there hasbeen no scientific follow-up of what actually happens with theintestinal flora, and the effect, if any, has mostly been uncertain,probably because no bacterial colonisation has taken place in theintestine or because the bacteria supplied lack the capacity to affectthe diarrhoea-inducing bacteria. Efforts have also been made to replacethe existing bacterial flora in the nasal mucous membrane, the skin andthe pharynx with harmless microorganisms, and it has been shown thatso-called recolonisation is possible and may be effective.

As mentioned above, supplying bacteria to patients suffering fromdifferent illnesses induced by pathogenic microorganisms has mostly beeninconclusive, obviously because of insufficient knowledge of whatbacteria strains are best suited for bringing about colonisation andinterference.

By working under given conditions, it has in recent years been possibleto establish important principles for the mechanisms controlling theinteraction between different microorganisms, and also to developmethods for examining this interaction. Thus, it has become possible tointervene in this interaction in a meaningful and reproducible fashion,which may be used therapeutically/prophylactically when treatinginfections.

SUMMARY OF THE INVENTION

The present invention relates to a preparation for controllingpathogenic microorganisms in man and in animals, with a view topreventing and/or treating diarrhoea and other gastrointestinaltroubles, said preparation being characterised by containing a viablemicroorganism strain in the form of Streptococcus lactis strain LIa inat least one pharmaceutically acceptable carrier medium in which themicroorganism retains its viability. Further characteristics of theinvention are apparent from the following text and the appended claims.

When studying the interaction between different bacteria, we haveconcentrated on the ability of certain lactic-acid bacteria to preventthe growth of pathogenic bacteria. On a laboratory scale, cultures ofStreptococcus lactis have been tested, and a large number of clones withdifferent biological properties have been isolated therefrom. Some ofthese have proved to have a pronounced inhibiting effect on most of theinfection-inducing bacteria in man, such as Stafylococcus aureus,various species of Salmonella, Shigella, Pseudomas, Klebsiella-Entero,and Campylobactus.

An important reason for the effectiveness of the Streptococcus lactisstrains is that some of these secrete bacteriocines which have aspecific killing effect on some invading microorganisms.

The inventive pharmaceutical preparation has a microorganism straincontent of 10⁶ -10¹⁰, preferably 10⁸ -10⁹, microorganisms/ml of finalpreparation ready for use.

In a preferred embodiment, the carrier for the microorganism strain isan acidulated or fermented milk product, such as soured milk, ropy milk,yoghourt, kefir and the like. The microorganism strain may be used asstarter culture when preparing such milk products, which results inacidulated or fermented milk products having an increased content of theadvantageous strain. Of course, it is also possible to add the bacteriato completed milk products, thereby obtaining a higher content in themedium of the desired bacteria. This embodiment is of considerableimportance, since the preparation obtained is a wholesome and popularfoodstuff. Such a preparation may be administered for prophylactic orcurative purposes against diarrhoea and other intestinal infectionsoften affecting infants. Further, it may be given to people who havereceived cancer therapy involving antibiotics or radiation, as well asto tourists travelling abroad where the bacterial flora is oftendifferent from that at home.

The preparation may also be in dry form for oral administration, e.g. ascapsules, tablets or powder. The carrier media used in this case are ofconventional type and well-known to the expert in the field. Thecapsules may be resistant to gastric juice, so that the bacteria areprotected therefrom and only released in the intestine. For the samereason, tablets may be provided with a known coating which is resistantto gastric juice. Further, powders may be stirred prior to use in anaqueous liquid, such as water, milk and fruit juice.

The preparation has proved to be a highly effective prophylactic againstvarious pathogenic intestinal bacteria causing gastrointestinaltroubles, such as "tourist diarrhoea".

Advantageously, the preparation may also be used for treatingSalmonella-infected people and animals.

Further, the preparation may also be adapted for treating animals, inwhich case the carrier is a suitable animal feed.

The microorganism should be kept in a freeze-dried state, preferably inskimmed milk in a dark and dry place, or frozen at a temperature ofabout -70° C.

The requisite dose of the inventive preparation is determined dependingon such factors as the age and general state of the patient, as well asthe type and seriousness of the illness. Anyone skilled in the art willhave the competence necessary for determining the suitable dose. Anotheradvantage of the invention is that a large overdose of thepharmaceutical preparation hardly involves any risks at all to thepatient.

THE MICROORGANISM OF THE INVENTION

The microorganism strain forming part of the inventive pharmaceuticalpreparation was isolated in the following manner. A naturally-occurringStreptococcus lactis strain was isolated from a steepgrass, and examinedfor its inhibitory effect on pathogenic intestinal bacteria. Sevenclones were tested, and one called LIa gave the best results.

This microorganism strain, which is used in the inventive preparation,was deposited in 1989 at The National Collection of Industrial andMarine Bacteria (NCIMB), Torry Research Station, P.O. Box 31, 135 AbbeyRoad, Aberdeen AB9 8DG, Great Britain. Its NCIMB accession number is40157. The microorganism strain is typed or classified according to theAPI 20 system (API System, La Balme les Grottes, 38390 Montalieu,Vercieu, France).

                                      TYPING TABLE                                __________________________________________________________________________    api 20 STREP                                                                                                   RESULTS                                      TESTS                                                                              SUBSTRATES    REACTIONS/ENZYMES                                                                           NEGATIVE       POSITIVE                      __________________________________________________________________________                                     VP 1 + VP 2/wait for 10 min                  VP   Pyruvate      Formation of acetoin                                                                        colourless     rose/red                                                       NIN/wait for 10 min                          HIP  Hippurate     Hydrolysis    colourless/pale blue                                                                         dark blue/violet                                               4 h    24 h    4 h       24 h                ESC  Esculin       β-glycosidase                                                                          colourless                                                                           colourless                                                                            grey black                                                                              black                                                pale yellow                                                                          pale yellow                                                                   light grey                                                             ZYM A + ZYM B/10 min (1)                                                      decolour with intense light if need be       PYRA Pyrrolidonyl-2-naphthyl                                                                     Pyrrolidonyl aryl amidase                                                                   colourless or very                                                                           orange                             amide                       pale orange                                  αGAL                                                                         6-bromo-2-naphthyl-α-D-                                                               α-galactosidase                                                                       colourless     violet                             galactopyranoside                                                        βGUR                                                                          naphthol-AS-BI-β-D-                                                                    β-glucuronidase                                                                        colourless     blue                               glucuronate                                                              βGAL                                                                          2-naphthyl-β-D-                                                                        β-galactosidase                                                                        colourless or very                                                                           violet                             galactopyranoside           pale violet                                  PAL  2-naphthyl-phosphate                                                                        Alkaline phosphatase                                                                        colourless or very                                                                           violet                                                         pale violet                                  LAP  L-leucine-2-naphthyl amide                                                                  Leucine aryl amidase                                                                        colourless     orange                         ##STR1##                                                                          Arginine      Arginine dihydrolase                                                                        yellow         red                                                            4 h    24 h    4 h       24 h                 ##STR2##                                                                          Ribose        Acidulation   red    orange/red                                                                            orange/yellow                                                                           yellow               ##STR3##                                                                          L-arabinose   Acidulation   red    orange/red                                                                            orange/yellow                                                                           yellow               ##STR4##                                                                          Mannitol      Acidulation   red    orange/red                                                                            orange/yellow                                                                           yellow               ##STR5##                                                                          Sorbitol      Acidulation   red    orange/red                                                                            orange/yellow                                                                           yellow               ##STR6##                                                                          Lactose       Acidulation   red    orange/red                                                                            orange/yellow                                                                           yellow               ##STR7##                                                                          Trehalose     Acidulation   red    orange/red                                                                            orange/yellow                                                                           yellow               ##STR8##                                                                          Inulin        Acidulation   red    orange/red                                                                            orange/yellow                                                                           yellow               ##STR9##                                                                          Raffinose     Acidulation   red    orange/red                                                                            orange/yellow                                                                           yellow               ##STR10##                                                                         Starch (2)    Acidulation   red    orange/red                                                                            orange/yellow                                                                           yellow               ##STR11##                                                                         Glycogen      Acidulation   red or orange  light yellow                  __________________________________________________________________________     (1) At a second reading after 24 h of incubation, a precipitate may be        noted in the tubes to which the reagents ZYM A and AYM B have been added.     This is a normal phenomenon, which can be ignored.                            (2) The acidulation of starch is often weaker than that of other sugars. 

    ______________________________________                                        TEST WITH API 20 STREP                                                        ______________________________________                                                VP             +                                                              HIP            +                                                              ESC            +                                                              PYRA           -                                                              αGAL     -                                                              βGUR      -                                                              βGAL      -                                                              PAL            -                                                              LAP            +                                                              ADH            +                                                              RIB            +                                                              ARA            -                                                              MAN            +                                                              SOR            -                                                              LAC            +                                                              TRE            +                                                              INV            -                                                              RAF            -                                                              AMD            -                                                              GLYG           -                                                              βHEM      -                                                      ______________________________________                                    

The strain has a weak α-hemolysis.

It grows well at 20°-37° C., and comparatively well at 39° C.

A suitable bouillon is M17, see recipe.

The strain belongs to the Lancefield N group.

MAKING SOURED MILK

In one embodiment of the invention, the pharmaceutically acceptablecarrier medium for the preparation is soured milk.

When implementing this embodiment, the microorganism LIa according tothe invention is allowed to grow in milk for 2-20 h, preferably 4 h.Then, the remaining soured-milk culture is added, which results infurther growth, but at a reduced temperature of 21° C., if thetemperature was higher at the beginning.

When the remaining soured-milk culture is added, the content of LIashould be 10⁶ -10¹⁰ cfu/ml, preferably 10⁸ -10⁹ cfu/ml. Then, the souredmilk is left for 20-24 h, preferably 20 h, whereupon it is cooled andfilled into containers.

After taking 260 ml of this soured milk in the morning and in theevening, the Streptocuccus lactis strain of the invention can be foundin the intestine even after 3-5 days.

We claim:
 1. A pharmaceutical composition for treating diarrhea cause bypathogenic gastrointestinal microorganisms comprising a viablemicroorganism strain of Streptococcus lactis strain LIa with NCIMBaccession number 40157 in a pharmaceutically acceptable carrier.
 2. Thepharmaceutical composition of claim 1, wherein the microorganism straincontent is 10⁶ -10¹⁰ microorganisms/ml.
 3. The pharmaceuticalcomposition of claim 2, wherein the microorganism strain content is 10⁸-10⁹ microorganisms/ml.
 4. The pharmaceutical composition of claim 1,wherein the pharmaceutically acceptable carrier medium is an acidulatedor fermented milk product.
 5. The pharmaceutical composition of claim 4,wherein the pharmaceutically acceptable carrier is an animal feed. 6.The pharmaceutical composition of claim 1, in a dry form.
 7. A method oftreating diarrhea caused by pathogenic gastrointestinal microorganismsin humans or animals comprising administering to a human or animal inneed thereof an effective amount of a pharmaceutical compositioncomprising a viable microorganism strain of Streptococcus lactis strainLIa with NCIMB accession number 40157 in a pharmaceutically acceptablecarrier.
 8. The pharmaceutical composition of claim 4 wherein theacidulated or fermented milk product is soured milk, ropy milk, yoghurtor kefir.
 9. The pharmaceutical composition of claim 6 wherein the dryform is a capsule, a tablet or a powder.